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Helix VIII of NhaA Na(+)/H(+) antiporter participates in the periplasmic cation passage and pH regulation of the antiporter
Authors:Diab Mohammad  Rimon Abraham  Tzubery Tzvi  Padan Etana
Institution:
  • Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel
  • Abstract:The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na+/H+ antiporter. However, because NhaA is active at physiological pH (pH 6.5-8.5), many questions related to the active state of NhaA have remained unanswered. Our Cys scanning of the highly conserved transmembrane VIII at physiological pH reveals that (1) the Cys replacement G230C significantly increases the apparent Km of the antiporter to both Na+ (10-fold) and Li+ (6-fold). (2) Variants G223C and G230C cause a drastic alkaline shift of the pH profile of NhaA by 1 pH unit. (3) Residues Gly223-Ala226 line a periplasmic funnel at physiological pH as they do at pH 4. Both were modified by membrane-impermeant negatively charged 2-sulfonatoethyl methanethiosulfonate and positively charged 2-(trimethyl ammonium)-ethylmethanethiosulfonate sulfhydryl reagents that could reach Cys replacements from the periplasm via water-filled funnels only, whereas other Cys replacements on helix VIII were not accessible/reactive to the reagents. (4) Remarkably, the modification of variant V224C by 2-sulfonatoethyl methanethiosulfonate or 2-(trimethyl ammonium)-ethylmethanethiosulfonate totally inhibited antiporter activity, while N-ethyl maleimide modification had a very small effect on NhaA activity. Hence, the size—rather than the chemical modification or the charge—of the larger reagents interferes with the passage of ions through the periplasmic funnel. Taken together, our results at physiological pH reveal that amino acid residues in transmembrane VIII contribute to the cation passage of NhaA and its pH regulation.
    Keywords:SH  sulfhydryl  TM  transmembrane  CL-NhaA  Cys-less NhaA  MTSES  2-sulfonatoethyl methanethiosulfonate  MTSET  2-(trimethyl ammonium)-ethylmethanethiosulfonate  MalNEt  N-ethyl maleimide  MTS  methanethiosulfonate  LBK  modified L broth  KPi  potassium phosphate
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